The goal of this research is to extend our knowledge of tRNA gene organization and expression in Escherichia coli. Specific tRNA genes which ave been isolated using recombinant DNA methodologies will be studied with regard to primary structure, then utilized for detailed studies on gene expression in vivo and in vitro. Specific tRNA leu regulatory sequences will be utilized in vitro transcription systems in order to determine what factors regulate tRNA gene expression. These controlling elements will be fused in vitro to the galactokinase gene of E. coli and utilized for detailed in vivo and in vitro studies thus simplifying physiological and genetic analysis. In vitro mutagenesis of these sequences will be employed to critically assess promoter and terminator structure-function relationships. This work impinges on the following major unanswered questions: How are all the tRNA genes of a cell coordinately expressed to ensure faithful protein synthesis? What are the general structural features of tRNA genes and their regulatory sequences? What factors are involved in the controlled initiation and termination of tRNA gene transcription? Ultimately these studies should permit the engineering of modified tRNA genes which will be useful in understanding how tRNA functions in the cell.